One-carbon organic compounds such as methane and methanol are found extensively in nature, and are utilized as carbon sources by bacteria classified as methanotrophs and methylotrophs. Methanotrophic bacteria include species in the genera Methylobacter, Methylomonas, Methylomicrobium, Methylococcus, Methylosinus, Methylocystis, Methylosphaera, Methylocaldum, and Methylocella (Lidstrom, 2006). Methanotrophs possess the enzyme methane monooxygenase, that incorporates an atom of oxygen from O2 into methane, forming methanol. All methanotrophs are obligate one-carbon utilizers, unable to use compounds containing carbon-carbon bonds. Methylotrophs, on the other hand, can also utilize more complex organic compounds, such as organic acids, higher alcohols, sugars, and the like. Thus, methylotrophic bacteria are facultative methylotrophs. Methylotrophic bacteria include species in the genera Methylobacterium, Hyphomicrobium, Methylophilus, Methylobacillus, Methylophaga, Aminobacter, Methylorhabdus, Methylopila, Methylosulfonomonas, Marinosulfonomonas, Paracoccus, Xanthobacter, Ancylobacter (also known as Microcyclus), Thiobacillus, Rhodopseudomonas, Rhodobacter, Acetobacter, Bacillus, Mycobacterium, Arthobacter, and Nocardia (Lidstrom, 2006).
Most methylotrophic bacteria of the genus Methylobacterium are pink-pigmented. They are conventionally referred to as PPFM bacteria, being pink-pigmented facultative methylotrophs. Green (2005, 2006) identified twelve validated species in the genus Methylobacterium, specifically M. aminovorans, M. chloromethanicum, M. dichloromethanicum, M. extorquens, M. fujisawaense, M. mesophilicum, M. organophilum, M. radiotolerans, M. rhodesianum, M. rhodinum, M. thiocyanatum, and M. zatmanii. However, M. nidulans is a nitrogen-fixing Methylobacterium that is not a PPFM (Sy et al., 2001). Methylobacterium are ubiquitous in nature, being found in soil, dust, fresh water, sediments, and leaf surfaces, as well as in industrial and clinical environments (Green, 2006).
The existence of PPFM bacteria as colonizers of the leaf surfaces of most (if not all) species of plants (ranging from algae, mosses and liverworts, and angiosperms and gymnosperms) suggests that PPFM bacteria may play an important role in plant physiology (Corpe and Rheem, 1989; Holland and Polacco, 1994; Holland, 1997; Kutschera, 2007). The fact that plants produce and excrete methanol, probably as a waste product of pectin metabolism in growing plant cell walls, suggested to these researchers that a symbiotic relationship exists, with the PPFM bacteria feeding on the plant-produced methanol and in turn providing positive benefits to the plants. The suggested benefits of PPFM bacteria on plant physiology include positive effects on nitrogen metabolism, seed germination, and stimulation of plant growth through the provision of PPFM-generated cytokinin plant hormones. The use of PPFM bacteria to improve plant growth, plant yield, seed germination, male fertility, and plant nutritional qualities has been disclosed in U.S. Pat. No. 5,512,069, U.S. Pat. No. 5,961,687, U.S. Pat. No. 6,174,837, U.S. Pat. No. 6,329,320, U.S. Pat. No. 7,435,878, and US Patent Application Pub. No. 2006/0228797. In addition, PPFM bacteria have been found to increase the yield of cultivated algae, suggesting their application to the production of algae-derived biofuels (US Patent Application Pub. No. 2011/0269219).
The broad application of Methylobacterium to row crops, vegetables, and other cultivated plants, as well as in the production of algae-based biofuels, would require the efficient and inexpensive cultivation of enormous quantities of Methylobacterium cultures. Other industrial applications of Methylobacterium may also benefit from efficient Methylobacterium production techniques. Such industrial applications include the use of Methylobacterium as environmental pollution indicators (as certain Methylobacterium can grow on soot) and as irradiation-quality-control monitors in the packaged food industries (as certain Methylobacterium exhibit high resistance to gamma-ray irradiation). Other industrial applications include the use of Methylobacterium to degrade environmental pollutants (U.S. Pat. No. 5,418,161, U.S. Pat. No. 5,487,834, U.S. Pat. No. 6,107,067, U.S. Pat. No. 7,214,509), to produce useful industrial compounds, polymeric precursors, or biopolymers (U.S. Pat. No. 5,236,930, U.S. Pat. No. 5,686,276, U.S. Pat. No. 6,107,067), and recombinant proteins (US Patent Application Pub. No. 20060234336).
However, various publications in the subject area of PPFM cultivation suggest that there are significant obstacles to overcome in order to achieve the efficient and inexpensive large-scale cultivation of these bacteria. Holland and Polacco (1994) reported that “isolated PPFMs do not grow well on plant tissue culture media”, a medium which is rich in nutrients, and that “PPFMs are slow growers”. Madhaiyan et al. (2004) state of PPFM bacteria that “Their slow-growing nature and distribution over the whole plant suggest that their numbers are regulated simply by dilution as the plant tissue expands away from growing points.” Abanda-Nkpwatt et al. (2006) reported of growth of PPFM bacteria that “in liquid culture, the solution became turbid within 4-5 days” without specifying the titer achieved (titer referring to the number of bacterial cells, or colony-forming units, per milliliter).
These consistent reports of slow growth are further confirmed and expanded upon by other studies indicating that PPFM bacteria could only be grown to relatively low titers. These growth studies were in standard liquid microbiological media, which are purposely prepared so as to be “water-clear”. Such media permit the visual observation and detection of both desired and undesired (i.e. contaminating) microbial growth, manifest as the development of turbidity visible to the naked eye.
Corpe and Basile (1982) presented a systematic investigation of the growth responses of various PPFM bacteria to a wide variety of carbon sources. They utilized as their base medium the standard mineral base employed by Stanier et al. (1966). In that publication, Stanier et al. stated of their base medium that “It is heavily chelated with nitriloacetic acid and EDTA, and forms a copious precipitate upon autoclaving. The precipitate redissolves as the medium cools, to form a water-clear solution.”
Using this “water-clear” solution as their base medium, Corpe and Basile (1982) tested a wide variety of carbon sources for their ability to support the growth of PPFM bacteria. They found several carbon sources that were relatively better than all the others, namely glycerol, glutamate, methanol, glucose, aspartate, succinate and malate. However, even after 7 days of incubation (the time allotted to each growth test), none of the cultures achieved an optical density (at 660 nanometers, the standard wavelength to measure microbial growth) of greater than 0.7 optical units, and most were well below this density. Sy et al. (2005) reported that a suspension of PPFM bacteria with an optical density of about 0.05 optical units contained about 5×106 colony forming units (CFU) of PPFM bacteria per milliliter. Thus, the maximum titer that Corpe and Basile achieved after one week of incubation with the best carbon sources they identified was about 7×107 colony-forming units per milliliter.
Sy et al. (2005) also reported that with a minimal salts medium containing succinate as the carbon source, they achieved a final titer of M. extorquens of about 2.5×108 colony-forming-units per milliliter.
Corpe and Rheem (1989) reported that PPFM bacteria “had a much longer doubling time than other leaf heterotrophs, in nutrient broth and other common heterotrophic media”, and concluded that methanol produced by plants “may allow the PPFMs to compete successfully” with other bacteria on leaf surfaces. The maximum titer that Corpe and Reehm achieved (after an unspecified incubation period) was about 3×108 colony-forming units per milliliter.
Thus, these publications indicate that in standard “water-clear” microbiological growth media, the growth of PPFM bacteria is slow and typically plateaus at a relatively low final titer of about 3×108 colony-forming units per milliliter.
In order to meet the potential needs for PPFM bacteria for commercial applications in row crops, vegetables, and other cultivated plants, as well as in the production of algae-based biofuels, manufacturing capabilities would need to produce enormous quantities of these bacteria.
Taking corn as just one example, there are about 40 million hectares of corn grown each year in the United States. For each 1% of market penetration (400,000 hectares) in this single nation and on this single crop, the need for PPFM bacteria can be estimated to be in the range of about 30 liters per hectare of PPFM culture with a titer of about 3×108 colony-forming units per milliliter, applied either as a seed treatment or as a foliar spray. This equates to about 12 million liters of PPFM culture at that titer being required each year to treat 1% of the United States corn crop. If the production time per batch was 7 days, a facility with even the largest volume fermenters on the market (producing 60,000 liters per batch) running at full capacity (about 250 days per year) would require 5 or 6 of these huge fermenters (again, just to supply the need for 1% market penetration of corn in the United States). Such a facility probably could not be built and operated in a commercially viable manner.
Thus, there exists a need for the development of efficient and inexpensive large-scale production of Methylobacterium. 